Toll-like receptor (TLR) signaling networks are crucial as the front line of defense against several microbial and viral pathogens. Upon exposures to viral and bacterial ligands, signaling of TLRs proceeds with two distinct pathways to release inflammatory cytokines; one involves a cytosolic adaptor protein Myeloid differentiation primary response 88 (MyD88) and another requires TRIF (Toll-interleukin-1-receptor domain-containing-adapter-inducing interferon-?). Overactivation of TLRs is associated with several inflammatory environmental diseases, such as sepsis, asthma, atherosclerosis and cancer. Recent studies show that external factors, such as cholesterol-lowering statin drugs and acute and prolonged exposure to alcohol, can considerably influence lipopolysaccharide (LPS)-induced TLR4 signaling thereby significantly impacting immune systems' reaction against pathogens. While some of the proximal signaling proteins that transduce signals from toll-like receptors (TLRs) into the cytosol have been defined and are under study as therapeutic targets for inflammatory diseases, many regulators remain unidentified. Very few studies have investigated the comprehensive role of the TLRs signaling network as it applies to MyD88-dependent and independent (TRIF) pathways following exposure to particular bacterial and viral ligands in combination with external and environmental factors such as alcohol and statin drugs. Discovery of new signal regulators in the TLR pathways over the past several years has unfortunately been constrained by the limitations of traditional biochemical and genetic approaches, generating a substantial research gap to be filled. This proposal will address this need for more complete identification and categorization of inflammatory-disease-causing TLR signals and decipher the molecular and pathological roles of TLRs in several inflammatory diseases. Aim 1 of this proposal will develop several novel mass spectrometry-cleavable cross-linking strategies and a related software tool for identification of large-scale protein to protein networks. Aim 2 will show applications of these crosslinkers for comprehensive profiling of innate immune signaling pathways of two cell surface TLRs, TLR4 and TLR2 after exposure to bacterial ligands and external factors, such as statin drugs and alcohol. These studies will provide critical insights on TLR signaling cascades and their pathogenesis mechanisms. This research also has the potential to reveal how these novel mechanisms may be targeted in therapeutic development for several infectious and inflammatory diseases.